Method for the production of coenzyme q

ABSTRACT

Coenzyme Q of the formula   WHEREIN N IS AN INTEGER FROM 5 TO 10 INCLUSIVE, IS PRODUCED IN GOOD YIELD BY INCUBATING HYDROCARBON-ASSIMILATING MICROORGANISM IN A CULTURE MEDIUM COMPRISING A CARBON SOURCE CONSISTING MAINLY OF HYDRO-CARBONS CONTAINING NOT LESS THAN 10 PERCENT (VOLUME/VOLUME) OF NORMAL PARAFFINS OF NINE TO 23 CARBON ATOMS, INCLUSIVE, AND RECOVERING THE COENZYME Q accumulated in the culture broth.

United States Patent Nakao et al.

[451 Apr. 25, 1972 [54] METHOD FOR THE PRODUCTION OF COENZYME Q [72]inventors: Yoshio Nakao, lbaraki; Mltsuzo Kuno, Suita; SaburoYamatodani, Minoo; lsuke lmada, lbaraki; Hiroshi Morimoto, Kobe,

[21] Appl. No.: 668,694

[30] Foreign Application Priority Data Sept. 17, 1966 Japan ..4l/6i555[52] US. Cl. ..l95/28 R [5 1] int. Cl. ..Cl2b 1/00 [58] Field of Search..195/28, 3 H, 3 R, 29; 260/396 [56] References Cited UNITED STATESPATENTS 3,219,543 11/1965 Douros et al. l95/28 3,222,258 12/1965 iizukaet al..... ....l95/3 H 3,308,035 3/1967 Douros 195/28 3,355,296 11/1967Perkins et al. ..l95/3 H OTHER PUBLICATIONS Arch. of Biochem. andBiophy. Vol. 89, pp. 318- 321 (i960) Coenzyme 0, page, A.C. et al.

Primary Examiner-A. Louis Monacell Assistant Examiner-Gary M. NathAttorney-Wenderoth, Lind 8: Ponack ABSTRACT Coenzyme Q of the formulaOH3O CH;

Y Omo- -(CH2-CH=CC 2)nH wherein n is an integer from 5 to 10 inclusive,is produced in good yield by, incubating hydrocarbon-assimilatingmicroorganism in a culture medium comprising a carbon source consistingmainly of hydrocarbons containing not less than 10 percent(volume/volume) of normal paraffins of nine to 23 carbon atoms,inclusive, and recovering the coenzyme Q'accur'riuTate'd in'theculfu'rbroth.

8 Claims, No Drawings METHOD FOR THE PRODUCTION OF COENZYME Q Thisinvention relates to a method for the production of coenzyme Q. Moreparticularly, this invention relates to a method for producing coenzymeQ of the formula wherein n is an integer from 5 to inclusive, which comprises inoculating a hydrocarbon-assimilating microorganism 1 It isknown that coenzyme Q of the formula (I), i.e., coenzyme Q coenzyme Qcoenzyme Q-,, coenzyme Q8, C0611 zyme Q and coenzyme Q are contained inthe cells of various kinds of microorganisms (throughout thespecification and the working examples the integer after the coenzyme Qdesignates the number of its isoprene unit of the side chain) and thatcoenzyme Q is useful, e.g., for the treatment of diabetes mellitus, forthe treatment of peptic ulcer and for preserving spermatozoa forartificial insemination of livestock. For example, it is reported thatcoenzyme Q, is obtained from Escherichia coli, coenzyme Q fromSaccharomyces cerevisiae, Saccharomyces ludwigii and Zygasaccharo mycesbarkeri, coenzyme Q, from Proteus vulgaris and Saccharomyces cerevisiae,coenzyme Q from Azotobacter vinelandii, Escherichia coli and Serratiamarcescens, coenzyme Q,,

from Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas putida andPenicillium chrysogenum, and coenzyme Q from Pseudomonas denitrificansand Neurospora crassa. However, their content in such microorganismcells is very small. Therefore, the hitherto-employed method forproducing coenzyme Q by incubating such microorganism(s) in a culturemedium and subjecting the resultant microorganism cells to extraction isnot industrially profitable.

It has long been an art desideratum to establish an industriallyprofitable method for producing coenzyme Q.

The present invention, fulfilling the said desideratum, and thusproviding a useful and industrially feasible process for producingcoenzyme Q, is based on the following new findings.

Among the microorganisms belonging to various genera,

there are included microorganisms which are capable of assimilatinghydrocarbons, especially normal paraffins having a carbon atom numberwithin the range from nine to 23 inclusive, which are readily availableat a relatively low cost as carbon sources from among various kinds ofhydrocarbons.

Further, when these microorganisms are incubated in a culture mediumwherein the carbon source consists mainly of hydrocarbons containing thesaid normal paraffins, a considerably large amount of coenzyme Q isaccumulated in the microorganism cells, and not infrequently aremarkably large amount of such cells is obtained, as compared with thecase of conducting incubation in a conventional medium containing ascarbon sources carbohydrates such as glucose and molasses. Thus,coenzyme Q can be recovered from the culture broth in a good yield at alow cost.

It is thus the main object of this invention to provide a new and usefulmethod for the industrial production of coenzyme Q Another object of theinvention is to-minimize troublesome procedures and disadvantagesentailed in effecting the hitherto-known microbial process for theproduction of coenzyme Q.

The microorganisms employable for the purpose of the present inventioncan be selected from those which can assimilate hydrocarbons, inaccordance with the kind of desired coenzyme O. For example, for thepurpose of producing coenzyme Q and/or coenzyme R there are employedmicroorganisms belonging to the genera Pseudomonas, Candida orAchromobacter. Such microorganisms are exemplified, e.g., by Pseudomonasalkanolytica nov. sp., Pseudomonas aeruginosa, Candida tropica'lis,Candida parapsilasis, Candida claussenii and Achramabacterparaflinoclastus nov. sp.

Among these microorganisms, Pseudomonas alkanolytica nov. sp. andAchromabacter paraffinoclastus nov. sp. were isolated from soil inOsaka, Japan and confirmed as novel specles.

The pseudomanas alkanolytica Nov. sp. has the following characteristicswhich are confirmed according to the description in the Manual ofMicrobiological Methods, Society of American Bacteriologists 1957):

1. Cell Characteristics:

Small rods near to cocci, 0.9 by 1.1 microns. Non-motile andnon-sporulating.

II. Staining characteristic:

Gram-negative.

lll. Cultural characteristics:

1. Nutrient agar plate: Untransparent greyish white, circular, smooth,entire, convex surface.

2. Nutrient agar slant: Growth abundant, greyish white spreading.

3. Nutrient agar stab: Growth on surface, filiform.

4. Nutrient broth: Turbidity with sediment, non-formation of velum. IV.Physiological characteristics:

1. pH relations: Growth at pH 5.0 to 9.0, optimum at about 7.0. 2.Temperature relations: Growth at 15 to 37 C, optimum at 37 C.

3. O relations: aerobic.

4. Gelatin: No liquefaction.

5. Starch assimilation: Negative.

6. Urea assimilation: Negative.

7. lndole: Not produced.

, 8. Ammonia: Not produced.

9. Hydrogen sulfide: Not produced.

10. Nitrates reduction: Nitrates produced from nitrates.

l l. Catalase: Positive 12. Acetylmethylcarbinol: Not produced.

13. Methyl red test: Negative.

l4. Litmus milk: Coagulated and acidic litmus not reduced.

15. Acid but no gas from glucose, galactose, mannose,

xylose. No acid and no gas from fructose, arabinose, sucrose, lactose,maltose, mannitol, sorbitol, glycerol.

Detailed comparison of the above-mentioned characteristicswith thedescriptions in Bergeys Manual of Determinative Bacteriol gy," SeventhEdition, reveals that this microorganism belongs to the genusPseudomonas. But this microorganism is quite different in microbialcharacteristics from any of the species belonging to Pseudomonasdescribed in the above-mentioned reference. Therefore, thismicroorganism is a novel species of the genus Pseudomonas, and has beennamed Pseudamonas alkanolytica.

A specimen of Pseudomonas alkanolyzica nov. sp. has been deposited atAmerican Type Culture Collection, Rockville, Maryland, under theaccession number ATCC 21034.

The Achramobacter paraffinaclastus nov. sp. has the followingcharacteristics which are confirmed according to the description in theabove-mentioned Manual of Microbiological Methods:

1. Cell characteristics:

Small rods, 0.8 to L2 by 1.0 to 2.0 microns. Non-motile andnon-sporulating.

ll. Staining characteristic:

Gram-negative.

Ill. Cultural characteristics: 7

1. Nutrient agar plate: Untransparent greyish white, circular, entire,convex surface.

2. Nutrient agar slant: Greyish white and filiform.

3. Nutrient agar stab: Growth on surface, filiform.

4. Nutrient broth: Slight turbidity with sediment, formation of thinvelum.

lV. Physiological characteristics:

1. pH relations: Growth at pH 5.0 to 9.0, optimum at about 2.Temperature relations: Growth at 15 to 37 C, optimum at 30 C.

3. O, relations: aerobic.

4. Gelatin: No liquefaction.

5. Starch assimilation: Negative.

6. Urea assimilation: Negative.

7. lndole: Not produced.

8. Ammonia: Produced.

9. Hydrogen sulfide: Produced.

l0. Nitrates reduction: Nitrites not produced from nitrates.

ll. Catalase: Positive.

12. Acetylmethylcarbinol: Not produced.

l3. Methyl red test: Negative.

l4. Litmus milk: Litmus slightly reduced.

15. No acid and no gas from glucose, fructose, galactose, mannose,xylose, arabinose, sucrose, lactose, maltose, mannitol, sorbitol,glycerol.

Detailed comparison of the above-mentioned characteristics with thedescription in Bergeys Manual of Determinative Bacteriology," SeventhEdition, reveals that this microorganism belongs to the genusAchromobacter judged from such characteristics as Gram-negative,non-motile, no production of soluble pigment, no assimilation ofglucose, no action to agar and no alkalization of litmus milk. But thepresent microorganism is quite different in characteristics from any ofthe species belonging to Achromobacter described in the said reference.Therefore, this microorganism is a novel species of the genusAchromobacter, and has been named Achromobacter paraffinoclastus.

A specimen of Achromobacter paraflinoclastus nov. sp. has been depositedat American Type Culture Collection, Rockville, Maryland, under theaccession number ATCC 21 130. 7

According to this invention, it is generally preferable to employ aliquid culture medium, and the incubation is carried out aerobically,i.e., with aeration and agitation either under static conditions or by asubmerged process. The culture medium employed in this invention isrequired to contain, as a carbon source, hydrocarbons containing normalparaffins of a carbon atom number within the range of nine to 23inclusive, especially 14 to 20 inclusive.

The amount of the said normal paraffins contained in the hydrocarbonsshould be not less than percent of the whole amount of hydrocarbonsemployed, from the standpoint of the growth of the microorganism as wellas the yield of the objective coenzyme Q.

The normal paraffms may consist of only one kind of normal paraffins ofa carbon atom number within the said range of nine to 23 or of two ormore kinds of normal paraffins, each having a carbon atom number withinthe aforesaid range of nine to 23. Hydrocarbons consisting only of thesaid normal paraffins are most advantageously employed in the presentinvention. But the hydrocarbon source in the present invention may,occasionally, contain other hydrocarbons (e.g., branched paraffins,olefins, cyclic paraffins, aromatic hydrocarbon, normal paraffins of oneto eight or not less than 24 carbon atoms, etc.) than the normalparafiins of carbon atom number within the aforesaid range of nine to23, as long as the normal paraffins of carbon atom number within thesaid range of nine to 23 are included as a whole in an amount of notless than 10 percent.

From the viewpoint of both the growth of the microorganism and yield ofthe objective coenzyme Q, the hydrocarbons are generally used in such anamount as to make the concentration of the normal parafiins of carbonatom number within the said range of nine to 23 in the culture medium,as a whole, about 3 to 15 percent, inclusive.

As these hydrocarbons are hardly soluble in water, the addition thereofto an aqueous culture medium is practically carried out under stirringor shaking to prepare a suspension containing then as very fineparticles. If desired, a suspending agent, e.g., a surfactant of thetype of polyoxethylene sorbitan monostearate (commercially available,e.g., as Tween-60) is employed.

in addition to the hydrocarbons, the culture medium advantageously alsocontains nitrogen source as a nutrient. As the nitrogen source, any ofthose used in the hitherto-known methods can be employed, and these areexemplified by inorganic nitrogen compounds such as ammonium salts,nitrates, etc. or nitrogen-containing materials such as peptone, soybeanpowder, comsteep liquor, meat extract, yeast extract, etc. Furthermore,a small quantity of inorganic salts such as sodium chloride, potassiumphosphate, salts of metals, e.g., of magnesium, zinc, iron, manganese,etc. may be added to the medium.

Incubation conditions such as the pH of the medium and the incubationtemperature are controlled so as to have the objective coenzyme Qaccumulated in maximum amount.

Generally, the pH of the culture medium and the incubation temperatureare respectively adjusted to about 5.0 to 9.0, preferably about 6.0 to8.0, and to about 28 to 37 C.

Under the above-mentioned culture conditions, the desired coenzyme Q isproduced and accumulated in the microorganism cells in the culturebroth.

Incubation is continued until the maximum amount of the objectivecoenzyme Q is accumulated in the microorganism cells. Although theperiod required for the maximum accumulation of the coenzyme Q ischangeable depending on various factors, the amount of coenzyme Q in themicroorganism cells reaches the maximum after 5 to 72 hours incubation.

The coenzyme Q thus accumulated in the cells is recovered by simpleprocedure. For example, the cells separated from the culture broth areeither (1) extracted with a suitable nonpolar organic solvent such ashexane, chloroform, isooctane or heptane, after saponification byheating with an alkali such as sodium hydroxide, potassium hydroxide,etc. or (2) extracted with a suitable hydrophilic organic solvent andthe resulting extract extracted with above-mentioned non-polar organicsolvent, if desired, after being subjected to saponification by heatingwith the said alkali or to treatment with an acid such as sulfuric acidor hydrochloric acid under heating. Thusobtained coenzyme Q-containingextract can be subjected to column chromatography with the use of asuitable adsorbent such as alumina, silicic acid, magnesium silicate andaluminum silicate to obtain the objective coenzyme Q as crystals.

Following examples are merely intended to illustrate presently preferredembodiments of the present invention and not to restrict the scope ofthe latter. Throughout the present specification as well as in thefollowing examples, the abbreviations pg, g" and C respectively refer tomicrogram(s), gram(s) and degrees centigrade. In the following examples,parts by weight bear the same relation to parts by volume as do grams tomilliliters. The coenzyme Q is determined by the method described inBiochimica et Biophysica Acta volume 32, page 73 (1959), afterseparation by the method appearing in Biochemische Zeitschrift volume336, page 380(1962).

EXAMPLE 1 TABLE 1 Normal paraffin mixture NPLCI I00 parts by volume 6parts by weight The mixture consists of normal parafl'lns of carbon atomnumbers within the range from 11 to 23, the specific normal paraffinsbeing contained in the following percentage relative to the whole weightof the mixture, and has a boiling range from about 262C to about 349C:

Carbon Atom Number Percent The resultant culture broth is inoculated in1,000,000 parts by volume of the culture medium of the same compositionas that in Table 1, and incubated with aeration and agitation at C for12 hours, the medium being kept at pH 7.0 with aqueous ammonia duringthe period of the incubation.

After the incubation, the resultant cells are collected bycentrifugation to give 74,000 parts by weight (in terms of dried cells)of cells s containing 1300 pg/g (on dry basis) of coenzyme Q and 818[Lg/g (on dry basis) ofcoenzyme Q The cells are suspended in 500,000parts by volume of ethanol. The suspension is kept standing at 25 C for14 hours, followed by heating at 60 C with agitation for 1 hour, thenthe suspension is centrifuged to separate the ethanol layer from thecells. The extraction of the cells in 500,000 parts by weight of ethanolat 60 C with agitation for 1 hour is repeated three times, and theethanol extracts are combined. The combined solution is subjected toextraction four times with hexane, each in a quantity of 300,000 partsby volume. Thus-obtained hexane solution is concentrated and dried underreduced pressure to give 8,830 parts by weight of residue.

The resultant residue is dissolved in 20,000 parts by volume of hexane,and the resulting solution is allowed to pass through a column packedwith 88,000 parts by weight of activated magnesium silicate(commercially available as Florisil; see also US. Pat. No. 2,393,625).The column is washed first with 1,200,000 parts by volume of hexane, andsubsequently with 1,100,000 parts by volume of hexane and chloroform(9:1 by volume). Then, the coenzyme Q adsorbed on the column is elutedwith 4,400,000 parts by volume of a mixture of hexane and chloroform(4:1 by volume). The coenzyme Q-containing solution is evaporated todryness under reduced pressure to give 133 parts by weight of residue.the residue is dissolved in 5,000 parts by volume of ethanol and thesolution is kept standing at 0 C to yield 65 parts by weight ofa mixturesubstantially consisting of coenzyme Q, and coenzyme Q,,.

Thus-obtained product is separable into each component, i.e., coenzymeQ, and coenzyme Q for example, by the following procedure.

The mixture is dissolved in 1,000 parts by volume of hexane and thesolution is chromatographed on two plates of silica gel treated withparaffin, using a mixture of acetone and water (9:1 by volume). Partscorresponding to coenzyme Q, and coenzyme O are eluted with 20,000 partsby volume of ethanol, respectively. Each extract is evaporated todryness under reduced pressure and the resultant residue is dissolved in500 parts by volume of hexane, then the solution is allowed to passthrough a column packed with 5,000 parts by weight of Florisil. Afterbeing washed with 50,000 parts of hexane, the column is eluted with50,000 parts by volume of a mixture of hexane and chloroform (1:1 byvolume), respectively. Each eluate is evaporated to dryness underreduced pressure, and the residue is dissolved in 2,000 parts ofethanol. Each solution is kept standing at 0 C overnight to give 42parts by weight of coenzyme Q, as yellow platy crystals melting at 42.5C and 20.5 parts by weight of coenzyme Q; as yellow platy crystalsmelting at 36 to 38 C.

In the process of this example, when the incubation process is carriedout with a culture medium containing parts by weight of glucose in placeof the normal paraffin mixture, the microorganism cannot substantiallygrow.

EXAMPLE 2 Achromobacter paraffinoclastus nov. sp. (ATCC 21130) isinoculated and incubated in 1,000,000 parts by volume of the culturemedium of the same composition as that in Table 1, with aeration andagitation at 30 C for 10 hours after the manner described in Example 1,whereby 62,000 parts by weight (in terms of dried cells) of cellscontaining 950 pg/g (on dry basis) of coenzyme Q and 560 tg/g (on drybasis) of coenzyme 0,, are obtained.

The cells are treated after the manner described in Example 1 to yield30 parts by weight of coenzyme Q, as yellow platy crystals and 18 partsby weight of coenzyme Q as yellow platy crystals.

1n the process of this example, when the incubation process is carriedout in a culture medium containing 100 parts by weight of glucose inplace of the normal paraffin mixture, the microorganism cannotsubstantially grow.

EXAMPLE 3 Pseudomonas aeruginosa (ATCC 21036) is incubated in 1,000,000parts by volume of the culture medium of the same composition as that inTable l with aeration and agitation at 30 C for 24 hours after themanner described in Example 1,

whereby 55,000 parts by weight (in terms of dried cells) of EXAMPLE 4Pseudomonas alkanolytica n0v.sp. (ATCC 21034) is incubated in 1,000,000parts by volume of the culture medium set forth in Table 2, withaeration and agitation at 30 C for 14 hours, the medium being kept at pH7.0 with aqueous ammonia, during the period of the incubation.

TABLE 2 Normal paraffin mixture 100 parts by volume Nl-LC] 6 parts byweight KH,PO 15 parts by weight Na l- P0 0.2 part by weight MgSO '7H O2.5 parts by weight CaC1 2H- O 0.5 part by weight FeSO '7l-l O 0.1 partby weight Yeast extract 2.0 parts by weight water up to 1,000 parts byvolume The mixture consists of normal paraffins of carbon atom numberswithin the range from 9 to 15, the specific normal paratfins beingcontained in the following percentage relative to the whole weight ofthe mixture, and has a boiling range from about 172 to 266C:

Carbon Atom Number Percent EXAMPLE 5 Candida tropicalis (ATCC 20026) isincubated in 1,000,000 parts by volume of the culture medium of the samecomposition as that in Table I with aeration and agitation at 28 C forhours after the manner described in Example 1, whereby 45,000 parts byweight (in terms of dried cells) of cells containing 750 ,ug/g (on drybasis) of coenzyme Q; is obtained.

The cells are treated after the manner described in Example I to yieldparts by weight of conenzyme Q, as yellow platy crystals.

In the process of this Example, when the incubation process is carriedout in a culture medium containing 100 parts by weight of glucose inplace of the normal paraffin mixture, less yield of coenzyme Q i.e., 7.5parts by weight of coenzyme Q,, is obtained.

EXAMPLE 6 Candida parapsilosis (ATCC lO232)is incubated in l,000,000parts by volume of the culture medium of the same composition as that inTable l with aeration and agitation at 28 C for 20 hours after themanner described in Example 1, whereby 40,000 parts by weight (in termsof dried cells) of cells containing 840 .tg/g (on dry basis) of coenzymeQ,, is obtained.

The cells are treated after the manner described in Example 1 to yield20 parts by weight of coenzyme Q as yellow platy crystals.

in the process of this Example, when the incubation is carried out in aculture medium containing 100 parts by weight of glucose in place of thenormal paraffin mixture, less yield of coenzyme 0,, i.e., 7.5 parts byweight of coenzyme Q, is obtained.

EXAMPLE 7 Candida claussenii (ATCC 20027)is incubated in 1,000,000 partsby volume of the culture medium of the same composition as that in Tablel with aeration and agitation at 28 C for 20 hours after the mannerdescribed in Example 1, whereby 48,000 parts by weight (in terms ofdried cells) of cells containing 870 rig/g (on dry basis) of coenzyme Q.is obtained.

The cells are treated after the manner described in Example 1 to yield28 parts by weight of coenzyme Q,.

In the process of this Example, when the incubation is carried out in aculture medium containing parts by weight of glucose in place of thenormal paraffin mixture, less yield of coenzyme Q9, i.e., 8.5 parts byweight of coenzyme Q, is obtained.

Having thus-disclosed the invention, what is claimed is:

l. A process for producing coenzyme Q of the formula wherein n is aninteger from 5 to 10 inclusive, which comprises incubating a coenzymeQ-producing microorganism selected from the group consisting of theCandida genus, and

Achromobacrer paraflinoclastus n ov. sp. A.T.C.C. 21130 in a culturemedium until the objective coenzyme Q is substantially accumulated inthe microorganism cells and recovering therefrom the coenzyme 0 soaccumulated, the microorganism being a hydrocarbon-assimilatingmicroorganism and the culture medium comprising a carbon sourceconsisting mainly of hydrocarbons containing not less than 10 percent(volume/volume) of normal paraffins having a carbon atom number withinthe range from nine to 23 inclusive.

2. A process according to claim 1, wherein the culture medium containsabout 3 to about 15 percent (volume/volume) of said normal parafiinsbased on the culture medium.

3. A process according to claim 1, wherein the normal parafiins have acarbon atom number within the range from 14 to 20 inclusive.

4. A process according to claim 1, wherein the hydrocarbon-assimilatingmicroorganism belongs to the genus Candida.

5. A process according to claim 1, wherein the hydrocarbon-assimilatingmicroorganism is Candida tropicalis.

6. A process according to claim Lwherein the hydrocarbon-assimilatingmicroorganism is Candida parapsilosis.

7. A process according to claim 1, wherein the hydrocarbon-assimilatingmicroorganism is Candida claussenii.

8. A process according to claim 1, wherein the hydrocarbon-assimilatingmicroorganism is Achromobacter paraffinoclastus nov. .rp. A.T.C.C. 21

2. A process according to claim 1, wherein the culture medium containsabout 3 to about 15 percent (volume/volume) of said normal paraffinsbased on the culture medium.
 3. A process according to claim 1, whereinthe normal paraffins have a carbon atom number within the range from 14to 20 inclusive.
 4. A process according to claim 1, wherein thehydrocarbon-assimilating microorganism belongs to the genus Candida. 5.A process according to claim 1, wherein the hydrocarbon-assimilatingmicroorganism is Candida tropicalis.
 6. A process according to claim 1,wherein the hydrocarbon-assimilating microorganism is Candidaparapsilosis.
 7. A process according to claim 1, wherein thehydrocarbon-assimilating microorganism is Candida claussenii.
 8. Aprocess according to claim 1, wherein the hydrocarbon-assimilatingmicroorganism is Achromobacter paraffinoclastus nov. sp. A.T.C.C. 21130